Most cells can be cultured in vitro to a limited extent using conventional cell culture technology, provided that suitable nutrients and other conditions for growth are supplied. Such cultures have been used to study genetic, physiological, and other phenomena, as well as to manufacture certain biomolecules using various fermentation techniques. In studies of mammalian cell biology, cell cultures derived from lymph nodes, muscle, connective tissue, kidney, dermis, and other tissue sources have been used, for example. However, most normal cells have a limited growth potential in culture. After a certain number of cell divisions (the Hayflick limit), they can no longer proliferate (Hayflick L., Exp. Cell. Res., 1965, 37:614–636). This limited life span, termed replicative senescence, likely arose as a protective mechanism against unfettered clonal evolution and cancer in long-lived animals. Therefore, while it has long been a goal of scientists to be able to maintain all types of cells in vitro, standard culture conditions do not promote the long-term survival or proliferation of most cells.
“Immortalization” is the escape from the normal limitation on growth of a finite number of division cycles. Therefore, once immortalized, a cell line can be continuously cultured. However, immortal cell lines very rarely emerge spontaneously under usual culture conditions.
In order to increase the life span of cells in culture, published techniques have included the use of embryonic cells. The strategy of starting with embryonic cells is based on the fact that embryonic cells are relatively less differentiated than adult cells, and thus can be expected to go through several cycles of cell division before becoming terminally differentiated. It is an axiom of biology that undifferentiated cells proliferate at a greater rate than differentiated cells. It is generally believed that by the time a cell has developed the necessary intra-cellular machinery for hormone synthesis and secretion, for example, it is no longer able to divide rapidly, if at all.
Another known strategy for establishing cells in culture is to start with tumor cells, due to their greater potential for proliferation. While these types of cell lines are able to generate a large number of cells, the limited number of these types of lines, the limited number of phenotypes that they are able to generate, and their inherent tumorgenicity, make these types of cell lines less than ideal.
Normal cells have been transformed in culture by various means including the use of UV light, chemical carcinogens, and the introduction of oncogenes, which alters the genetic programming of the cell, thereby inducing the cell to proliferate indefinitely. Simian virus 40 (SV40) has been used for some time to immortalize human cells from different tissues in order to gain continuously growing cell lines (Sack, G. H. In vitro, 1981, 17:1–19). Rat granulose cells were transformed by co-transfection with the entire SV40 genome and the activated Ha-ras gene (Baum, G. et al. Develop Biol, 1990, 112:115–128). These cells were reported to retain at least some differentiated characteristics, i.e., they were able to synthesize steroids in response to cAMP. It has also been shown that expression of SV40 large T protein alone is sufficient to induce transformed properties in primary cells (Abcouver, S. Bio/Technology, 1989, 7:939–946). Other cell lines established in culture include UMR cells, derived from normal islets of neonatal rats (NG, K. W. et al., J. Endocrinol., 1987, 113:8–10) and HIT cells, derived by SV40 infection of hamster islets (Santerre, R. F. et al., PNAS, 1981, 78:4339–4343). The insulin secretory output of these cell lines is low, however, and response to glucose is lost with passage in culture. Thus, while the proliferative status of these cell lines may prove useful for studying the decisions that occur during cell determination and differentiation, and for testing the effects of exogenous agents, these immortalization agents may affect other properties of the cell, such as the cell's ability to differentiate and express genes in a physiologically correct manner.
More recent methods of cell line immortalization that are still in the beginning stages of development involve telomeres, the ends of chromosomes composed of non-coding repeat DNA sequences. It has been suggested that the limited reproductive lifespan of normal (diploid) cells in culture may be explained by an inevitable shortening of one or more telomeres. It is known that cancer cells, germ cells, and some eucaryotic microorganisms have the ability to correct this phenomenon with the enzyme telomerase, which catalyzes telomere elongation. Normal cells modified to express telomerase are immortal in culture (Bodnar et al., Science, 1998, 279(5349):349–352), presumably by maintaining a constant telomere length. Furthermore, in vitro-aged fibroblasts treated with telomerase regain dermal function (Funk et al., Exp. Cell. Res., 2000, 258(2):270–278).
Only a few neuronal cell types have been reported to divide in the adult brain and adult neurons do not survive well in vitro. The generation of clonal cell lines from different regions of the brain would greatly facilitate the discovery of new neurotrophic factors and their receptors, and enhance the understanding of their function. The central nervous system contains two major classes of cells known as neurons and glial cells. There are hundreds of different types of neurons and many different neurotrophic factors that influence their growth and differentiation. Depending upon the type of neuron and the region of the brain in which the neuron resides, a different neurotrophic factor or specific combination of factors affect the survival, proliferation, and differentiation of the neuron.
To date, neuropharmacological studies in the central nervous system (CNS) have been delayed by the lack of cell systems needed to investigate potentially useful neuroactive compounds. In live animals, the complexity of the brain makes it difficult to effectively measure which cellular receptors are being targeted by these compounds. Additionally, the expense involved in live animal research and the current controversies stemming from animal rights movements have made in vivo animal studies less acceptable for initial research. Primary cells from neuronal tissue are often used for CNS studies; however, long-term culture of primary neurons has not been achieved. Only a few attempts to achieve long term culture and proliferation of neuronal cells have been reported. In fact, the proliferation of neuronal cells has proven so elusive that it has become ingrained in the scientific community that neuronal cells do not proliferate in vitro. As a consequence, fresh dissections must be performed for each study in order to obtain the necessary neuronal cell types, resulting in costly research with increased variability in the experimental results.
While some neuronal tumor cell lines exist, they are few in number and are not well characterized. In general, these tumor cell lines do not mimic the biology of the primary neurons from which they were originally established. In vitro primary cultures that are more phenotypically representative of primary cells and that could generate continuous cultures of specific neuronal cell lines capable of proliferation would be invaluable.
Similar to neurons, the endocrine cells of the mammalian pancreas have been considered to be post-mitotic, i.e., terminal, essentially non-dividing cells. Recent work has shown that the cells of the mammalian pancreas (including those of humans) are capable of survival in culture, but are not capable of sustained cell division. Hence, a primary culture of the tissue cells can succeed, but due to a lack of sufficient cell divisions of the cultured cells, passaging of the primary culture to form serial cultures has not been possible. Although occasional cells in a metaphase stage, uptake of tritiated thymidine, and other evidence of cell division have been seen in these cultures (Clark et al., Endocrinology, 1990, 126:1895; Brelijie et al., Endocrinology, 1991, 128:45), the overall rate of cell division has been considered to be below the replacement rate (that is, more, or as many, cells die as are produced).
The culture of animal cells in vitro, as “biofactories,” for the production of various proteins, peptides, hormones, growth factors, and other biologically active substances has been widely investigated. For example, pituitary cells have been cultured in vitro to produce growth hormone; kidney cells have been cultured to produce plasminogen activator; and hepatitis-A antigen has been produced in cultured liver cells. Other cells have been specifically cultured to produce various viral vaccines and antibodies. Interferon, insulin, angiogenic factor, fibronectin and numerous other biomolecules have been produced by the in vitro culture of various animal cells. Of course, the quantity of biomolecules produced by these biological factories is limited by the numbers of cells and range of cell types available.
Various cell lines have also been used in animal models of transplantation for a variety of purposes. Fetal kidney cells and amniotic cells have been transplanted as sources of trophic factors. Adrenal medullary cells, sympathetic ganglion cells, and carotid body cells have been transplanted as sources of dopamine. Fibroblasts and glial cells have been transplanted as sources of trophic factors, to carry genes through recombinant strategies, or for demyelinating diseases, for example. Corneal endothelial cells have been used for corneal transplants. Myoblasts have been transplanted for the treatment of muscular dystrophy and cardiac disease. Other cell lines include pancreatic islet cells for diabetes; thyroid cells for thyroid disorders; blood cells for AIDS, bone marrow transplant, and inherited disorders; bone and cartilage for osteoarthritis, rheumatoid arthritis, or for fracture repair; skin or fat cells for reconstructive purposes, such as in skin grafts after burns or cosmetic surgery; breast augmentation with fat; hair follicle replacement; liver cells for liver disorders inducing hepatitis; and retinal pigment epithelial cells (RPE) for retinitis pigmentosa and Parkinson's disease.
Unfortunately, the inability to procure large numbers of primary cells that are genetically stable has impeded the ability of medical science to progress in the area of cell transplant therapy. In addition, current sources for therapeutic donor cells are limited further by the inherent biological variability among the donors.
Stem cells are believed to have immense potential for therapeutic purposes for numerous diseases. Stem cells have been derived from numerous donor sources, including, but not limited to, embryonic, blast, tissue-derived, blood, and cord-blood cells; organ-derived progenitor cells; and bone marrow stromal cells; among others. Such stem cells can be differentiated along numerous pathways to produce virtually any cell type. These cells can be transplanted either before or after differentiation. From a therapeutic perspective alone, such cells may be useful for the treatment of a vast array of disorders. Examples of neurological disorders that can potentially be treated with stem cells include Parkinson's disease, Alzheimer's and Huntington's diseases, ALS, stroke, demyelinating disorders, epilepsy, head trauma, and spinal cord injury. However, stem cells share the same problem with other cells relating to the ability to proliferate the cells in vitro in sufficient quantities for diagnostic, investigational, or therapeutic purposes. Moreover, primary stem cells that have exhibited the most plasticity are embryonic stem cells. Obtaining large quantities of these cells is particularly problematic and raises ethical issues.
The above description of the state-of-the-art makes it apparent that there is a need for methods to maintain any and all cells in long-term cultures at increased proliferation rates, thereby providing a more plentiful and less costly supply of cells. Such long-term cultures could be developed as biological “factories” for the production of therapeutically useful proteins, for example. Well-established cell lines would also offer the possibility of in vitro bioassays based on the cells' responses to drugs and other chemicals (e.g., for toxicity and efficacy studies). There is also a need for the ability to produce a homogenous cell line, particularly a homogenous cell line of human origin. The availability of cells and cell lines that can be cryo-preserved is likewise lacking.
Continuously cultured cell lines would also be invaluable as a source of cells for cell transplant therapy, which has been found effective in correcting many disease states. For instance, diabetics could be stabilized and possibly cured through the implantation of cells that replace the function of insulin-secreting β-cells of the pancreas. Parkinson's patients could be treated with a ready supply of dopaminergic neurons, or stem cells giving rise to dopaminergic neurons. Such cell lines would also provide an endless supply of cells and tissue readily accessible for genetic modulation in vitro prior to transplant, for use in cell-mediated gene therapy. Thus, there exists a need for methods to produce cells and cell lines that would proliferate for extended periods in vitro yet faithfully retain their differentiated functions.